The authors have nothing to disclose.

All procedures including human subjects have been approved by Dharmais Cancer Hospital Ethics Committee with certificate number of 040/KEPK/VII/2017. All patients signed the consent forms and expressed agreement to participate in this study.
NOTE: The inclusion criteria are patients with diagnosis of early breast cancer, with tumor stage T1-T2 without palpable and ultrasonography lymph nodes enlargement (cNo). The exclusion criteria are locally advanced breast cancer, received neo adjuvant chemo or hormonal therapy, and pregnancy.
1. Preparation of 1% methylene blue dye and the injection technique
<ol>
	<li>Sterilize the surgical field after anesthetizing the patient.</li>
	<li>Aspirate 2 mL of 1% methylene blue dye from its vial with a 3 mL syringe.</li>
	<li>Draw a line to mark the lower axillary hairline below the lateral border of pectoralis major muscle.</li>
	<li>Inject 2 mL of 1% methylene blue dye into the subareolar space of the breast.</li>
	<li>If there is a scar in the upper outer quadrant of the breast or NAC, inject 2 mL of 1% methylene blue dye peritumorally into the breast parenchyma at the lateral side of the scar towards the axilla with a 23G needle under ultrasound guidance with a linear probe (12 MHz).</li>
	<li>Massage the breast circularly at the injection site for 5 min, and then continue to perform surgery.</li>
</ol>
2. Sentinel node biopsy technique in breast conserving surgery (BCS)
NOTE: The surgery is performed in a patient who underwent BCS and SNB.
<ol>
	<li>Prepare the surgical tools: monopolar electrocautery, DeBakey forceps/anatomical forceps, and retractors.</li>
	<li>Incise the skin, subcutaneous tissue, and fascia.</li>
	<li>Find the blue nodes or blue lymphatic tracts. Follow the blue tracts until the blue nodes or non-blue nodes with lymphatic blue tracts are identifiable.</li>
	<li>Search for the sentinel nodes along the intercostobrachial nerve and lateral thoracic vein if the blue nodes or blue lymphatic tracts cannot be found.</li>
	<li>Resect the sentinel nodes carefully and avoid damaging the nodes.</li>
	<li>Palpate the axillary space to find additional suspicious malignant lymph node enlargement.</li>
</ol>
3. Sentinel node biopsy technique in mastectomy
NOTE: The surgery is done in a patient who underwent mastectomy and SNB.
<ol>
	<li>Incise the skin and subcutaneous tissue.</li>
	<li>Create skin flaps.</li>
	<li>Remove the breast from pectoralis major until axillary fossa can be fully exposed.</li>
	<li>Incise the clavicopectoral fascia to find the sentinel node.</li>
	<li>If the blue lymphatic tracts cannot be found, find the sentinel node along the intercostobrachial nerve and lateral thoracic vein area.</li>
	<li>Remove the sentinel node.</li>
	<li>Look for additional suspicious lymph nodes by palpation.</li>
</ol>
4. Intraoperative examination
<ol>
	<li>Slice the lymph nodes no thicker than 2 mm, parallel to the long axis.</li>
	<li>Make touch imprint cytology from each node.</li>
	<li>Place the surgical specimen on a metal tissue disc and embed in a gel-like medium with the same density as frozen tissue.</li>
	<li>Submit all of the nodes for frozen section (FS) examination.</li>
	<li>Categorize the metastatic status of sentinel nodes into positive or negative, and report it to the surgeon during the surgery.</li>
</ol>
5. Pathological examination
<ol>
	<li>Perform the final pathologic evaluation of the sentinel nodes on formalin-fixed and paraffin-embedded tissue sections.</li>
	<li>Classify the sentinel nodes metastasis according to the 6th edition of American Joint Committee on Cancer (AJCC) manual. Macrometastasis (MAC) is defined as tumor deposits larger than 2 mm, micrometastasis (MiC) is defined as tumor deposits between 0.2 and 2 mm, isolated tumor cells (ITC) are defined as cell clusters no larger than 0.2 mm.</li>
	<li>Perform the serial sectioning and immunohistochemistry analysis for cytokeratin when there are doubts over defining ITC.</li>
	<li>Examine the rest of axillary lymph nodes in a similar manner.</li>
</ol>

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We thank Muhammad, M.D. for preparing and assisting the surgery, Mr. Ali Abdul Aziz, Mr. Adhitya Bayu, and Mr. Ariananda Hariadi in helping with the manuscript preparation. We also acknowledge Mr. Tegar Kharisma for his help on video editing.

In the modern era of breast cancer surgery, SNB has replaced ALND as the standard of care for axillary staging in early breast cancer and ALND should be abandoned if the SNs are free from metastasis14,15. The lymphatic mapping technique which is commonly used in developed countries is the application of radioisotope tracer and PBD as a combined or single technique16,17. The question on how to perform SNB ...

The status of axillary lymph nodes (ALNs) metastasis is the most important prognostic factor in breast cancer. Axillary lymph node dissection (ALND) was the conventional procedure to assess metastatic status of ALNs1,2. Unfortunately, ALND results in morbidities to the patients which decreases the quality of life, especially by increasing the risk of lymphedema after this procedure3,4. Nowadays, sentinel node biopsy (SNB) has replaced ALND for axillary staging because of its minimal morbidities among patients5. The most common method for performing SNB is using radioisotope tracer and PBD6. In some parts of the world, including in developing countries, these tracer agents could be difficult to procure and searching for an alternative tracer agent is critical to solve the problem.
Initially, MBD was used by Wong et al. as a tracer agent for mapping sentinel nodes (SNs)7. In their study using a feline model, intradermal injection of MBD showed poor lymphatic uptake and isosulfan blue was chosen as the preferred dye for sentinel node (SN) mapping7. Methylene blue dye has been used in breast cancer SNB since the first successful report by Simmons et al.8. Several studies have also reported MBD as the favorable dye for SNs identification, and that the false negative rates of MBD technique were comparable to radioisotope or PBD9,10,11. Fewer allergic reactions and lower price are the other reasons to consider its use in SN mapping12.
Recently, we studied the use of 1% MBD alone for SNB in clinically node negative breast cancer. In early stages, MBD has a favorable identification rate and negative predictive value13. We inject 2 mL of 1% MBD into the subareolar space or peritumoral area if there was an excisional biopsy scar at the upper outer quadrant or nipple areolar complex (NAC) of the breast. The blue nodes and non-blue nodes with lymphatic blue channels are categorized as SNs. The anatomical landmark in the axilla is used as a guidance to find SNs. Intraoperative examination is applied to assess the metastasis and the SNs are sent for histopathology analysis based on American Society of Clinical Oncology (ASCO) Guidelines14.
If the cases are selected carefully and the skills required for this technique are obtained by the surgeons as well as pathologists, many patients could be saved from the harmful effects of ALND while still having favorable survival.

<table border="0" cellpadding="0" cellspacing="0" style="width:1046px;" width="1046">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:348px;">Metiblo 50mg/5ml</td>
			<td style="width:174px;">Laboratories Sterop NV</td>
			<td style="width:353px;">BE475217</td>
			<td style="width:171px;">Methylene Blue Dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable syringe with needle - 3mL (Luer Lock Tip)</td>
			<td>Terumo Europe NV</td>
			<td>dvr-3414</td>
			<td>Syringe for Injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ForceTriad energy platform</td>
			<td>Medtronic</td>
			<td>ForceTriad</td>
			<td>Surgical cautery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shandon Cryomatrix embedding resin</td>
			<td>Thermo (scientific)</td>
			<td>6769006</td>
			<td rowspan="2">Frozen Section</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cryotome FSE</td>
			<td>Thermo (scientific)</td>
			<td>77210153</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HistoStar Embedding Workstation</td>
			<td>Thermo (scientific)</td>
			<td>A81000001</td>
			<td rowspan="3" style="width:171px;">Histopathological Examination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Finesse Me+</td>
			<td>Thermo (scientific)</td>
			<td>A77510272</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gemini AS</td>
			<td>Thermo (scientific)</td>
			<td>A81500002</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Benchmark GX</td>
			<td>Ventana Medical Systems</td>
			<td>750-850</td>
			<td rowspan="2">Immunohistochemistry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Benchmark XT</td>
			<td>Ventana Medical Systems</td>
			<td>750-700</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Olympus</td>
			<td>Model BX53</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ultrasound</td>
			<td>Phillips</td>
			<td>HD 7XE</td>
			<td></td>
		</tr>
	</tbody>
</table>

the application of 1% methylene blue dye as a single technique in breast cancer sentinel node biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

Here, we describe the results from the presented technique. Two milliliters of 1% MBD were injected at the deep subareolar space as the standard technique of injection (Figure 1A). If the peritumoral injection is indicated, it should be performed under ultrasound guidance (Figure 1B). The blue nodes or lymphatic blue tracts were seen after entering the axillary space. Following the lymphatic blue tracts lead to finding the SNs (<...

Watch this Scientific Journal Video about The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy at JoVE.com

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

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JoVE Journal

Cancer Research

18.8K Views.  Dharmais Cancer Hospital. In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs). 

Video: The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...